In this competitive renewal grant, we propose to continue moving forward our research program toward our long-term objective to understand at a molecular level how the signaling phospholipid PIP2 controls the activity of diverse ion channels. In the last cycle of this research program, based on available crystal structures, we developed 3-D models of Kir channels, such as Kir2.1, docked 4 PIP2 molecules onto the model , and guided by our mutagenesis data, chose the size of the system to perform Molecular Dynamics (MD) simulations. Such simulations predicted mostly interacting residues that had been previously implicated in PIP2 sensitivity by mutagenesis work and additional residues that still need to be tested experimentally. Our simulations revealed the Na coordination site in the CD-loop of Kir channels and guided us to identify a similar coordination site in the diverse Slo2.2 channel. Moreover, comparing simulations with and without PIP2 revealed explicit changes in networks of interactions that stabilize either the closed (in the absence of PIP2) or open (in the presence of PIP2) state. We have experimentally probed one such network by mutagenesis of specific interactions and the results support strongly the predictions of our theoretical model. We propose to perform a comprehensive study of key changes caused by the presence of PIP2 in the mammalian Kir2.2 channel, whose structure was recently solved. MD simulations of Kir2.2 with and without PIP2 reveal key roles for stabilization of two of the three channel gates in the closed or open states: the slide helix, B-loop and CD-loop stabilizes the G-loop gate; while the pore helix, and the two transmembrane domains stabilize the selectivity filter gate. Mutagenesis analysis will explicitly test these predictions and guide further simulations to elucidate the role of specific mutants. In parallel, as part of the current grant we have shown experimentally that PIP2 stabilizes the closed state of the gate controlled by the voltage sensor, while it also stabilizes an open state of another yet non- identified gate. Borrowing from what we have learned from Kir channels, we propose a similar analysis on Kv1.2, where MD simulations with and without PIP2 will guide experiments to elucidate how PIP2 controls Kv channel gates in this complex way. Relevance Pursuing the molecular mechanism by which PIP2 controls channel gating and voltage sensitivity, using two recently determined ion channel structures, will further our biophysical understanding of three diverse channels and potentially help with drug design for cardiac arrhythmias. PUBLIC HEALTH RELEVANCE: PIP2 has surprised us all in the last dozen years, as it has been found to control the activity of most ion channels it has been tested on. Signaling the hydrolysis of PIP2, in other words taking PIP2 away from the channel, can cause channel inhibition for those channels that interact with this phospholipid with moderate or weak affinity. Although we have made good progress in identifying both experimentally and computationally the channel residues interacting with PIP2 (previous funding cycles), we still do not know how such interactions control specific channel gates. In this proposal we are seeking to develop powerful predictive structural models of how PIP2 controls the activity of two diverse K+ channels, the Kir2.2 and Kv1.2, whose structures were recently determined. Because these channels require PIP2 for normal activity, obtaining a structural understanding of how PIP2 regulates their activity is potentially a powerful step toward designing drugs for cardiac arrhythmias.